The exon-1 of the androgen receptor (AR) gene contains two repeat length polymorphisms which modify either the amount of AR protein inside the cell (GGNn, polyglycine) or its transcriptional activity (CAGn, polyglutamine). Shorter CAG and/or GGN repeats provide stronger androgen signalling and vice versa. To test the hypothesis that CAG and GGN repeat AR polymorphisms affect muscle mass and various variables of muscular strength phenotype traits, the length of CAG and GGN repeats was determined by PCR and fragment analysis and confirmed by DNA sequencing of selected samples in 282 men (28.6±7.6 years). Individuals were grouped as CAG short (CAGS) if harbouring repeat lengths of ≤21 and CAG long (CAGL) if CAG >21. GGN was considered short (GGNS) or long (GGNL) if GGN ≤23 or >23, respectively. No significant differences in lean body mass or fitness were observed between the CAGS and CAGL groups, or between GGNS and GGNL groups, but a trend for a correlation was found for the GGN repeat and lean mass of the extremities (r=−0.11, p=0.06). In summary, the lengths of CAG and GGN repeat of the AR gene do not appear to influence lean mass or fitness in young men.
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Muscle mass and strength, as well as aerobic fitness (VO2max) are related to health and mortality.1 Muscle mass and strength is determined by environmental factors, principally endocrine, nutritional and mechanical loading, and by the genetic background.2 Gene polymorphisms, like those encoding for the insulin-like growth factor-1 (IGF-1),3 type I collagen (COL1A1),4 ciliary neurotrophic factor (CNTF),5 interleukin-6 (IL-6),6 the vitamin D receptor (VDR),7 IGF-2,8 resistin (RETN)9 and androgen receptor (AR),10 have an influence on either muscle mass or strength.
The AR gene is located to the X chromosome (q11.2–q12), and contains eight exons. The exon 1 contains a polyglutamine tract encoded by CAG repeats and a polyglycine tract (GGN) encoded by (GGT)3GGG(GGT)2(GGC)n. Polymorphic tracts are close to the region encoding the transactivation-1 domain of the AR protein.11 The CAG and GGN polymorphisms of the AR gene are related to incidence of prostatic cancer, breast cancer, plasma hormone levels and other metabolic, cardiovascular and even mental diseases.12,–,15
The polyglutamine repeat has an average length of 22 amino acids (range: 8–35). Short CAG repeats are associated with increased AR transactivation activity and stronger transcriptional potential.16 The CAG polymorphisms are associated with the fat-free mass phenotype in healthy elders.10 However, it remains to be established if the AR polymorphism influences muscle mass and fitness in young adults.
The polyglycine repeat length of AR ranges from 10 to 30.17 Short GGN repeats are associated with increased AR protein content in cell cultures that may in turn enhance the response to androgen stimulation.18 It remains unknown if a short GGN repeat number is associated to increased muscle mass or strength in humans.
The aim of this study was to determine if AR polymorphisms are associated to muscle mass and physical fitness in adult men. We tested the hypothesis of whether men with short CAG and/or short GGN repeats have greater fat-free mass and muscle mass, and, therefore, greater strength and muscle power, than those harbouring long CAG and/or long GGN repeats. Since studies in cell culture and animal models have shown that androgen–AR signalling pathway increases the expression of slow-twitch-specific skeletal muscle proteins leading to a more oxidative phenotype,19 we also studied whether AR polymorphisms have an effect on aerobic power (VO2max) in humans. This information may be useful to elaborate genetic profiles like those recently proposed by Lucia et al20,–,22 to explain individual variations in human physical performance.
Two-hundred and eighty-two Caucasian men participated in the study. They were recruited from physically active university students, sports clubs and local police officers in Gran Canaria (Spain). Recruitment started in February 2003 and extended to June 2007. The health status of each participant was established by a medical history and physical examination. Subjects taking any kind of medications or having any chronic disease or hypertension were excluded. The study was performed in accordance with the Helsinki Declaration of 1975 as regards the conduct of clinical research, being approved by the Ethical Committee of the University of Las Palmas de Gran Canaria. All volunteers provided their written informed consent before participation in the study.
Tests were carried out over 4 days. The first testing day started with a 20-ml blood sample which was obtained from an antecubital vein in the supine position, between 7:30 and 8:30. Body composition, jumping performance and maximal isometric force was tested on the second day. The last 2 days were used to assess sprint performance and anaerobic capacity, as well as maximal aerobic power (VO2max).
Whole body composition was assessed by dual-energy x-ray absorptiometry (DXA; QDR-1500; Hologic Corp., software version 7.10, Waltham, Massachusetts, USA) as reported in Perez-Gomez et al.23 Upper and lower limb lean mass (kg) was calculated from the regional analysis of the whole body scan,24 25 which gives a valid and reliable estimate of muscle mass in the extremities.26
Vertical jump performance and running sprint tests
The forces generated during vertical jumps were measured with a force platform (Kistler, Winterthur, Switzerland), as reported in Ara et al.27 Two kinds of jumps were performed: squat jump, in which countermovement was not permitted, and countermovement jump, from standing position subjects were asked to perform a countermovement, intending to reach knee bending angles of around 90° just before impulsion.
Subjects performed three maximal indoor 30-m running sprint trials to assess running speed with photocells (General ASDE, Valencia, Spain). Each sprint was separated by at least 5-min rest, and the best performance was recorded.28
An all-out 300-m running test was used to estimate the anaerobic capacity, since the anaerobic metabolic pathways contribute more than 50% to the overall energy expenditure during all-out exercise tests with a duration between 30 and 60 s.29 The test was performed on a 400-m track; the time was recorded manually with a digital stopwatch.
Aerobic maximal power
The maximal oxygen uptake (VO2max) was estimated using the maximal multistage 20-m shuttle run.30 The time during which the subjects were able to run for was recorded to calculate VO2max.
CAG and GGN repeat polymorphisms
DNA was extracted from blood samples (200 µl) using High Pure PCR Template Preparation Kits (Roche Applied Science). To determine the length of the CAG and GGN repeats, the corresponding regions located on the exon 1 of the AR gene (Genbank accession no. M27423) were amplified using two pairs of primers whose sequences have been previously reported.13 One primer from each pair was marked with fluorescent dye (FAM or VIC). Amplification was performed in a 25-µl reaction volume, containing 50 ng of genomic DNA, 200 µM of each deoxynucleotide triphosphate, 1× FastStart Taq DNA polymerase Buffer (Roche Applied Science, Mannheim, Germany), 1× GC-rich solution buffer (Roche Applied Science) and 1 U of FastStart Taq DNA polymerase (Roche Applied Science). The concentration of each pair of primers was 1.2 and 1.5 µM for the amplification of the CAG and GGN repeats, respectively. PCR conditions were 30 cycles of 95°C for 45 s, 56°C for 30 s and 72°C for 30 s for CAG amplification; 30 cycles of 95°C for 1 min, 55°C for 2 min and 72°C for 2 min for GGN amplification. Each PCR was initiated with a denaturation step at 95°C for 5 min and terminated with an extension step at 72°C for 5 min. The PCR product was diluted 1:100 in distilled water, and 1 µl of the dilution was mixed with 10 µl of formamide and 0.3 µl of GeneScan 500 LIZ Size Standard (Applied Biosystems, Warrington, UK), denatured at 98°C for 5 min and cooled on ice. Fragment separation was performed by automated capillary electrophoresis, using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems), and the length was determined with GeneScan Analysis Software (version 3.7; Applied Biosystems). Internal standards supplied by the manufacturer were used for quality control. We blindly repeated the genotype analysis in 54 of the samples, and the results were completely coincident. The fragments size was confirmed by sequencing 48 DNA samples harbouring different size alleles for both repeats by using the BigDye Terminator Sequencing Kit (Applied Biosystem) at University of Las Palmas Sequencing Facility. Genotyping was performed specifically for research purposes based on the hypothesis that the aforementioned polymorphisms may influence VO2max, lean mass and muscle strength. The genotype data of the subjects were not previously analysed for other non-research purposes and as such were not presented a posteriori for the present paper. The researchers in charge of genotyping were totally blinded to the subjects' identities, that is, blood samples were tracked solely with code numbers, and personal identities were only made available to the main study researcher who was not involved in actual genotyping.
All variables were checked for normal distribution by the Kolmogorov–Smirnov test. When necessary, the analysis was done on logarithmically transformed data. The influence of CAG and GGN repeat lengths on body composition and fitness was determined taking CAG and GGN repeat lengths as either continuous variables or as dichotomous variables with allele cut-off thresholds. The relationship between CAG and GGN as continuous variables with lean body mass and physical fitness variables was examined using linear regression analysis. The median value which resulted in the most balanced grouping was used as cut-off threshold. Thus, individuals were grouped as CAG short (CAGS; n=151) if harbouring repeat lengths ≤21 and CAG long (CAGL; n=131) if harbouring repeat lengths >21. Subjects were ascribed to the GGN short (GGNS; n=170) group if harbouring repeat lengths of ≤23; otherwise, they were included in the GGN long (GGNL; n=112) group. In addition, the subjects were also grouped if having any of the following haplotype combinations: CAGS+GGNL (n=64), CAGL+GGNS (n=83), CAGL+GGNL (n=48) and CAGS+GGNS (n=87).
Mean values were compared using analysis of variance with two factors (CAG and GGN lengths), each with two levels (short and long repeat number). Pairwise comparisons were tested for statistical significance using the Bonferroni post hoc test. Lean mass was corrected for differences in height by dividing muscle mass by height.2 31
Subject's body composition, anthropometrics, physical activity and fitness are reported in table 1. The observed allele frequencies for AR CAG and GGN repeat numbers in the studied subjects are presented in fig. 1. There were 17 different CAG alleles (ranging from 13 to 35 repeats) and 14 GGN alleles, ranging from 12 to 28 repeats.
CAG repeat polymorphism
Subject's body composition, anthropometrics, physical activity and fitness in the GGNS and GGNL groups are reported in table 2. The CAG polymorphism was not associated to any studied variable. No significant differences were found either in lean body mass or fitness between the CAGS and CAGL groups (table 2). There was no relationship between the length of the CAG repeat polymorphism and lean mass or physical fitness variables.
GGN repeat polymorphism
Subject's body composition, anthropometrics, physical activity and fitness in the GGNS and GGNL groups are reported in table 3. A trend for a significant inverse association between the logarithm of the length of the GGN polymorphism and the muscle mass of the extremities (MME) expressed as kg/height2 was observed (MME=11.6–2.3×Lg GGN, R=0.11, p=0.06). The length of the GGN repeat polymorphism did not correlate with any of the physical fitness variables assessed.
Interaction between CAG and GGN repeat polymorphism
The body composition, anthropometrics, physical activity and fitness of men grouped as CAGL+GGNL, CAGS+GGNS, CAGS+GGNL and CAGL+GGNS are reported in table 4. Although men having the combination CAGS and GGNS jumped 9.0% higher than those having the combination CAGL and GGNL (table 4), this effect was not significant after accounting for multiple comparisons (p=0.13). Differences between allele combinations in other physical fitness and lean mass variables were not significant, even without accounting for multiple comparisons.
This study shows that in physically active young men, AR polyglycine and polyglutamine repeat polymorphisms have no influence on lean mass or fitness when studied alone. Although the subjects with the combination CAGS and GGNS jumped higher than those with the combination of CAGL and GGNL, this effect disappeared after accounting for multiple comparisons. However, we cannot rule out a potential type II error, implying that this effect needs to be verified in future studies.
In agreement with previous studies, we did not observe any association between height and length of the CAG repeat polymorphism in men.10 Although the subjects having a CAG repeat number >22 had a 1.1% greater height2-adjusted lean body mass than the group with shorter alleles, this difference did not reach statistical significance. This is in contradiction to results from Walsh et al10 that reported a 2% greater lean body mass in the subjects with a CAG repeat number ≥22, in a group of 294 men with a mean age of 73 years. The difference between both studies is likely due to the fact that our subjects were much younger (29 years old) and had more appendicular muscle mass but lower height2-adjusted whole body lean mass than the subjects studied by Walsh et al, suggesting that with ageing men may increase trunk lean mass, due to changes in other components of the trunk lean mass apart from the muscle tissue as demonstrated by using potassium whole body counting.32 No significant differences in lean body mass, height or fitness were observed in men between the CAGS and CAGL groups. Like Walsh et al,10 we did not observe significant differences in appendicular muscle mass in men related to CAG repeats.
In the present investigation, we have also examined the influence of the GGN repeat AR polymorphism on muscle mass and physical fitness in healthy men. In agreement, with our hypothesis, there was a trend for an inverse relationship between the length of the GGN polymorphism and lean mass.
The possibility for an interaction between the CAG and GGN repeat polymorphism has not been previously studied. We have observed that the men having the microsatellite combination CAGS+GGNS could jump higher than those harbouring the combination CAGL+GGNL. The CAGS+GGNS microsatellite combination has been associated to stronger androgen signalling11 14; however, our results do not give clear support to the hypothesis that this combination may have a favourable influence in power-generating capacity of the skeletal muscles. Jumping performance is determined by body composition33 and factors that determine the speed of muscle activation and rate of force development, among which is critical the percentage of type II fibres.34 Performance in the 300-m running test depends not only in great part on the muscle mass35 but also on other factors which determine the contractile and metabolic properties of the muscles, such as a high percentage of fast-twitch (or type II) fibres and a high anaerobic capacity.29 36 In theory, the combination of CAGS+GGNS may confer a functional advantage for tasks requiring muscle power, but additional studies are required to clarify this point.
It remains to be elucidated if CAGS and/or GGNS haplotypes are associated to an increased proportion of type II fibres and/or enhanced anaerobic capacity. Animal studies indicate that increased androgen signalling may stimulate the expression of slow-twitch-specific skeletal muscle proteins while inhibiting fast-twitch-specific skeletal muscle proteins.19 However, there are no sex differences in muscle fibre types in humans,37 and 20 weeks treatment with testosterone enanthate did not change muscle fibres in men.38
Take home message
The length of CAG and GGN repeat of the AR gene do not appear to influence lean mass or fitness in young men. Additional studies are required to test if men harbouring the combination CAGS and GGNS have more jumping capacity.
The authors thank José Navarro de Tuero for his excellent technical assistance.
Competing interests None.
Funding The study was supported by Ministerio de Educación y Ciencia (DEP2006-56076-C06-04/ACTI) and FEDER, Consejería de Educación, Cultura y Deportes del Gobierno de Canarias (2006/179 0001 and FEDER), Proyecto Interreg IIIB BIOPOLIS, Fundación del Instituto Canario de Investigación del Cáncer (FICIC), Cabildo de Gran Canaria, Cabildo de Tenerife and La Caja de Canarias, and Proyecto Estructurante “Integración de los grupos de investigación en Ciencias de la Salud entorno al estudio de la obesidad y el síndrome metabólico con un enfoque molecular, celular, fisiopatológico, epidemiológico y psicosocial”, ULPGC, Gobierno de Canarias.
Ethics approval This study was conducted with the approval of the University of Las Palmas de Gran Canaria.
Provenance and peer review Not commissioned; externally peer reviewed.
Patient consent Obtained.